EDGE FGT: Development of functional genomics tools in cactophilic Drosophila

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The genome of organisms can have thousands to tens of thousands of genes coded in their DNA. For each gene, organisms can also produce alternative versions of a gene product (usually a protein) as well as being turned on in specific tissues or at specific times during development. Therefore, just possessing knowledge of the DNA sequence of an organism’s genome is not enough to understand the function of all its genes. To facilitate this, investigators over the past several decades have been able to manipulate or edit the genome of organisms using a variety of molecular tools. Unfortunately, these tools have traditionally only been available for a very selected group of laboratory model organisms. Although these model organisms have dramatically expanded our understanding of the function of genes during development, in specific tissues and sometimes all the way down to specific cell types, the limited number laboratory model species and generalist ecology has prevented the analysis of the function of genes in a comparative, evolutionary and ecological context. This project will leverage the extensive ecological knowledge of several cactus-breeding fly species in the genus Drosophila with their recently determined genome sequences to build a set of genome-manipulable strains. Using these modified strains of closely related species investigators will be able to assess the function of genes (e.g. turning them on/off in specific tissues/cells). Knowledge of this new toolkit and its application will be disseminated via publications, a public website with detailed protocols and videos as well as an annual workshop.The central aim of this project is to enhance the power of investigators to assess the relationship between genotypes and phenotypes in an ecological and comparative context by creating a customizable transgenic system. Here investigator Matzkin proposes to build a transgenic toolkit for the existing diverse community utilizing the ecological model system of cactophilic Drosophila. This system offers a unique opportunity to integrate genomics, functional genomics, transcriptomics and phenomics in an ecological and comparative context. Cactophilic Drosophila are endemic to the Americas and oviposit, develop, and feed as adults in the necrotic tissues of a variety of cactus species. The transgenic toolkit will be built using the three most widely used cactophiles, D. mojavensis and D. arizonae from North America and D. buzzatii from South America; using the recently generated de novo chromosome level genome assemblies and annotations of each of the four D. mojavensis cactus host populations, of two ecologically divergent D. arizonae populations and a new comprehensive annotation of D. buzzatii. To build the toolkit PI Matzkin will: 1) generate lines with attP integration sites in several chromosomes using CRISPR; 2) use these lines to incorporate C31 integrase in the X chromosome of all three species; 3) perform detailed tissue, developmental and environment-dependent transcriptional analysis to identify driver loci; 4) generate tissue specific GAL4 driver lines and a UAS-GFP reporter; 5) generate a UAS-RNAi plasmid construct; and 6) disseminate the toolkit using the National Drosophila Species Stock Center, a dedicated online repository for information (cactusflybase) and in-person workshops.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.